Analytical and diagnostic determinations can be performed on liquid samples by means of optical assays based on the detection of analytes in a sample, such as e.g. polynucleotide analytes, receptor proteins or antiligand molecules. One important application of optical assays is the field of immunology, in which the analyte is detected with the aid of a specific antibody, which is capable of binding to the analyte to form optically detectable complexes, e.g. by labeling the analyte with a fluorophore, or by providing a fluorophore-labeled conjugate before the optical detection. The detection may be performed by means of an optical reader, which is capable of illuminating the assay support substrate with an exciting light source and of detecting the fluorescent light emitted from the fluorophores.
An optical assay is performed by an optical assay arrangement comprising a sample supporting substrate and an optical reader. The optical reader comprises a source and detector for electromagnetic radiation within the optical wavelength region (i.e. between approximately 40 nm and 1 mm), and suitable optical waveguiding means for focusing and filtering. The sample support comprises a substrate of e.g. a polymeric material provided with one or more reaction site areas, comprising spots or lines of probe molecules, e.g. of an antibody, providing binding sites for molecules of the analyte, i.e. the target molecules, that may be present in a sample. When the sample is brought in contact with the probe molecules on the support surface, the target molecules in the sample will react with the probe molecules on the surface of the substrate and form optically detectable spots or lines. Luminescent light will be emitted when the substrate is illuminated with the exciting light source of the optical reader, thereby indicating that a reaction has occurred between the target molecules of the sample and the probe molecules of the reaction sites.
Luminescent light is emitted either as fluorescent light or phosphorescent light, or as chemiluminescent light. Fluorescence and phosphorescence may be defined as the emission of electromagnetic radiation resulting from absorbed exciting electromagnetic radiation, the fluorescent light lasting less than 1×10−8 s after the excitation, and phosphorescent light lasting longer, i.e. is decaying more slowly after the exposure to the exciting light, while chemiluminiscence is the emission of light resulting from a chemical reaction.
In fluorescence (and phosphorescence), the exciting radiation normally has a shorter wavelength (i.e. higher energy) than the emitted radiation, although the reverse may be true for multi-photon fluorescence. The fluorescent behaviour may be studied in a steady state or time-resolved, and fluorescence spectroscopy involves e.g. single- and multi-photon fluorescence, FRET (fluorescence resonance energy transfer), and fluorescence up-conversion. In fluorescence assays, the wavelength of the exciting and the emitted radiation depends on the type of fluorophore, which may be of an organic or inorganic origin, e.g. cyanine dyes or nanocrystals. As an example, the common fluorophore Cy5 is typically excited with 649 nm, and the emitted light is measured at 670 nm.
In optical assays, the concentration of an analyte in the sample may be determined by measuring the intensity of emitted fluorescent or phosphorescent light, by means of the light detector of an optical reader, thereby enabling quantitative measurements. Consequently, the efficiency of the illumination of a reaction site area with exciting light, as well as the efficiency of the collection of the emitted light, will have an effect on he performance of the optical assay.
Further, the reaction sites on a substrate surface may be provided with an array of spots or lines of different probe molecules, binding different target molecules. Therefore, an optical reader may be designed to be capable of determining the presence of several analytes in a sample, by means of different fluorophores.
A scanning optical reader is a conventional optical reading device for illuminating detection sites on a substrate and for detecting the emitted light. The scanning optical reader preferably comprises a narrowband exciting light source, such as a laser, and the laser light is focused on each individual detection site. The emitted light from each detection site is focused on an optical detector, such as a photodiode or a PMT (photomultiplier tube). In a scanning optical reader, the entire surface of the support substrate is scanned by a relative X-Y-movement between the optical means and support. The focusing means of a scanning optical reader may e.g. comprise confocal optics only collecting emitted light in the depth of focus of the objective lens, e.g. by blocking unwanted light by a pinhole and thereby reducing the detected noise.
An imaging optical reader is another conventional optical reading device, which is capable of detecting a two-dimensional array of pixels. The imaging optical reader comprises an exciting light source, e.g. a xenon lamp, for illuminating a large part of the surface area (or the entire surface area) of the substrate, and a detector capable of detecting emitted light from the entire detection site-area simultaneously, e.g. a CCD (Charged-Coupled Device)-imager, which utilizes MOS (Metal-On-Semiconductor)-technology, and offers a high quantum efficiency, sensitivity and spatial resolution. Further, a wideband light source may be provided with wavelength filters to provide monochromatic radiation.
A prior art sample supporting substrate for liquid samples is disclosed in WO 03/103835, said substrate being microstructured to form a capillary flow path for the sample, forming a pattern of micropillars protruding from the surface of the substrate. The size of the micropillars is in the micrometer-range, 1 micrometer (μm)=1×10−6 m, and the spacing is adapted to induce capillary action of the liquid. In an optical assay arrangement comprising a sample supporting substrate provided with micropillars, the light paths of both the exciting light and the emitted light will be affected by the micropillars, thereby influencing the performance of the optical assay arrangement. The optical properties of the material of the substrate, such as the light transmission, will also affect the optical assay performance.
A prior art optical reader is described in WO 01/575501, which discloses optical imaging of a sample on a transparent substrate, without any protruding microstructures. The optical reader comprises an exciting energy source to stimulate emission of detectable light from the sample, and the substrate is provided with a reflective surface located below the sample to reflect the emitted light into the detection means.
Further, a prior art optical assay arrangement is disclosed in WO 2004/104585, which relates to a microstructured microarray support, in which the height of the microstructures is adapted to the depth of focus of the optical arrangement.
It is an object of this invention to provide an optical reader and a polymeric sample substrate suitable for an optical assay arrangement, in order to achieve an improved optical assay performance, by accomplishing an efficient illumination of the reaction site area of the substrate and an efficient collection and detection of the emitted light, as well as a reduction of the detected optical background signal, thereby increasing the resulting signal-to-noise ratio.